The broad, long-term objective of this proposal is to isolate and characterize novel desaturase and acetylenase genes from the Basidiomycete fungi. The pursuit of valuable oilseed crops with altered oil compositions, unusual fatty acid components, or increased fungal resistance will be facilitated by the well-conceived manipulation of unsaturated and acetylenic fatty acid metabolism supported by our larger objective. Bioactive, acetylenic secondary metabolites with potential economic value include antimicrobial compounds, HIV reverse-transcriptase inhibitors, and antiproliferative agents. Unsaturated fatty acid biosynthesis has been implicated in plant phytoalexin production, a defensive response triggered by fungal pathogenesis. Despite the clear economic significance, most details of the enzymatic reactions required for the formation of acetylenic natural products remain elusive. In fact, no desaturase or acetylenase enzymes from entire phylum Basidiomycota, which include a very large proportion of acetylene-producing organisms and most fungi involved in wood decay, have been characterized to date. The specific aims of the proposal are: (1) to isolate acetylenase and desaturase genes from Basidiomycete fungi and (2) to characterize the activities of heterologously over expressed diverged desaturases and selected site-directed mutants. A delta-12 desaturase sequence mined from the White Rot Fungal Genome project will be isolated and characterized. Using the sequence of this putative linoleate-producing desaturase, existing and new Basidiomycete cDNA libraries for acetylene-producing fungi will be screened for homologs of the fungal and plant FAD2 oleate desaturase genes. The adaptation of plant microsomal desaturase preparations for use with fungal diverged-desaturase-expressing yeast microsomes will provide an added foundation for future biochemical studies. Comparison of the recently identified plant and the new fungal acetylenases/desaturases discovered through the proposed research will help delineate factors that control desaturase-like reactions in plants and macrofungi. Through the above studies, we will test the hypothesis that multiple FAD2-like enzymes are required for polyacetylene biosynthesis, while characterizing an understudied group of important fungal enzymes.